Free radicals mediate numerous physiological and pathophysiological processes including, but not limited to, aging, cancer, atherosclerosis, neurodegenerative diseases, cardiovascular diseases and diabetes. Free radicals are atomic or molecular species with unpaired electrons or an otherwise open shell configuration. The unpaired electrons are usually highly reactive, so free radicals are likely to take part in numerous chemical reactions.
Analyzing free radicals in biological samples/systems, however, has traditionally been challenging because free radicals are highly reactive entities with very short lifetimes. One method for analyzing free radicals is spin trapping coupled with electron paramagnetic resonance (EPR). Janzen E & Blackburn B, J. Am. Chem. Soc. 90:5909-5910 (1968). Spin trapping is based on a specific reaction between spin traps and free radicals that forms a paramagnetic spin adduct, which is less reactive than the free radicals, and thus accumulates in higher concentrations. Because spin adducts are paramagnetic, their EPR spectra provide information on the trapped free radical. Unfortunately, bioreduction and/or biooxidation of spin adducts can occur in biological applications of spin trapping. In addition, free radicals are often compartmentalized in biological samples/systems, and therefore not easily accessible for analysis.
More recent spin trapping methods utilize nitrone compounds that react with a target free radical to form a persistent and distinguishable spin adduct that can be detected by EPR spectroscopy. See, e.g., Fréjaville C, et al., J. Chem. Soc., Chem. Commun. 1793-1794 (1994); Fréjaville C, et al., J. Med. Chem. 38:258-265 (1995); Olive G, et al., Free Rad. Biol. Med. 28:403-408 (2000); Ouari O, et al., J. Org. Chem. 64:3554-3556 (1999); and Zeghdaoui A, et al., J. Chem. Soc. Perkin Trans. 2:2087-2089 (1995). These methods, however, each present its own set of limitations, which commonly include short persistency of the spin adducts, slow spin trapping kinetics, complicated spectra because of a mixture of the spin adducts and anisotropy of the signal when proteins are trapped. Consequently, identification of the spin adducts can be difficult.
One application of spin traps is to analyze mechanisms of protein S-nitrosation, which is a common NO-dependent, post-translational modification involved in numerous signaling pathways. While it is relatively straight forward to measure the total level of protein S-nitrosation using reductive chemiluminescence techniques (Samouilov A & Zweier J, Anal. Biochem. 258:322-330 (1998); and Zhang Y & Hogg N, Am. J. Physiol. Lung Cell Mol. Physiol. 287:L467-L474 (2004)), its detection of specific proteins currently relies on an indirect technique that involves a specific reduction of a S-nitroso group by ascorbate, followed by labeling of a newly formed thiol with a biotin label (Jaffrey S, et al., Nat. Cell Biol. 3:193-197 (2001)) or a fluorescent probe (Kettenhofen N, et al., J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 851:152-159 (2007)) in so-called ‘switch’ assays.
The reaction between ascorbate and S-nitrosothiols in switch assays, however, is not kinetically facile, and often times much higher levels of ascorbate have been used in this assay than originally proposed. In addition, the reaction lacks specificity, as numerous low molecular weight disulfides and other proteins have been shown to be reduced at significant rates under high-ascorbate conditions, leading to false positives. Moreover, several extracellular proteins, including serum albumin, result in positive signals with the biotin switch assay, but not with the chemiluminescence switch assay.
In addition to protein S-nitrosation, spin traps are used to analyze other protein free radicals, as well as lipid and nucleic acid free radicals.
Hence, there is a need for spin trapping compounds that trap free radicals and form persistent, detectable spin adducts that can be directly assessed by a variety of known methods. In addition, there is a need for spin trapping compounds that not only trap free radicals, but also can be targeted to an organ, a cell, an organelle or a molecule of interest.